Partial characterisation of pilchard herpesvirus and the associated disease in pilchards

Transcription

1 Partial characterisation of pilchard herpesvirus and the associated disease in pilchards This thesis is presented for the degree of Doctor of Philosophy at Murdoch University by Melanie Crockford 2007

2 Declaration The experiments in this thesis constitute work carried out by the candidate unless otherwise stated. The thesis is less than 100,000 words in length, exclusive of tables, figures, bibliography and appendices, and complies with the stipulations set out for the degree of Doctor of Philosophy by Murdoch University, Western Australia.. Melanie Crockford i

3 Abstract In 1995 and again in 1998, millions of pilchards (Sardinops sagax neopilchardus) were found dead or dying off the coast of Australia and also in New Zealand. The epizootics moved progressively, at a rapid speed against the prevailing currents. A previously unrecognised herpesvirus, Pilchard herpesvirus (PHV), was identified as the causative agent. Until recently, rapid and sensitive methods to detect PHV were not available and based on a previously identified and conserved 373 bp region of the genome, polymerase chain reaction (PCR), in situ hybridisation and real-time PCR methods were developed for the specific detection of PHV in formaldehyde-fixed and frozen tissues of pilchards. Real-time PCR was shown to have greater sensitivity than a conventional PCR and in situ hybridisation for the detection of PHV infection. The PCR assay and sequence analysis of the amplification products was used to compare the 373 bp region of the genome from strains obtained during the 1995 and 1998 epidemics. Significant differences between the strains were not detected. Additional sequence data was obtained adjacent to the 373 bp of known PHV sequence, which did not match any sequence in any of the genetic databases, and this will be invaluable for further study of the pilchard herpesvirus and the development of improved detection methods. The molecular-based methods of virus detection developed were applied to a reexamination of virus in paraffin-embedded tissues taken from fish during an attempt to transmit the virus to wild caught pilchards in The results obtained confirmed previously equivocal results that transmission of PHV to wild caught pilchards was achieved, although this experiment failed to demonstrate that transmission of the virus resulted in severe lesions typical of those seen in the epizootics. Using formaldehyde-fixed samples from fish collected during the 1998 PHV epizootic, virus was detected in fish collected 4 days prior to the occurrence of the epizootic even though the fish then appeared clinically normal, during the epizootic, and 8 days after mortalities had ceased. An investigation of wild pilchards collected from 4 Australian pilchard subpopulations using real-time PCR demonstrated that PHV was present in the gills of 13.75% of 800 fish sampled, indicating that the virus is now endemic in the Australian pilchard population. Variation in the prevalence of PHV infection in the 4 ii

4 subpopulations was detected, higher in western and southern populations than in populations from the east coast. The endemic nature of PHV infection in the pilchard population explains why there have been no further epizootics with mass mortalities since iii

5 Acknowledgements Work on Pilchard herpesvirus has been made possible through the support of the Fisheries Research and Development Corporation, who funded the project, the Department of Fisheries WA, where the majority of the work was done, and Murdoch University, WA. This project would not have been possible without mentoring from my supervisors, Dr. Brian Jones and Prof. Graham Wilcox, both of whom allowed me to tap into their wealth of knowledge. They assisted me in deciding upon aims and objectives, interpretation of data and when, where and how to use different writing styles. Thank you to Brian for his never-ending support and guidance throughout this project. For someone that is so busy, Brian has always made time to answer my questions and set me on the right path. Thank you also to Graham for his guidance, especially with data interpretation and in the editing of this thesis. This project allowed me to work with some interesting and talented people, but I would especially like to thank Dr. Mark Crane and Dr. Ken McColl of the Australian Animal Health Laboratory in Geelong, and also Prof. Richard Whittington of the University of Sydney for their hospitality during my visit, their guidance and contributions to work on Pilchard herpesvirus, and for broadening my scientific perspective. Thank you to my partner, Aaron, for enduring the many highs and lows of research, and the moods that go with them. I am sure that at times it felt like the world evolved around pilchards and herpesviruses, and that often during this project you didn t have my full attention. Our weekends can now be free of pilchards. And last, but certainly not least, to my dad, Graham, whose courage and strength in everyday life has taught me the power of persistence, determination and laughter. iv

6 Publications Published Crockford M. (2004) Australian and New Zealand Standard Diagnostic Procedure: White Spot Disease. PDF file on website: Crockford M., Jones J.B., Crane M.S.J. and Wilcox G.E. (2005) Molecular detection of a virus, Pilchard herpesvirus, associated with epizootics in Australasian pilchards Sardinops sagax neopilchardus. Diseases of Aquatic Organisms 68(1): 1-5 Jones J.B., Crockford M., Whittington R.J., Crane M.S.J. and Wilcox G.E. (2006) Aquatic Animal Health Subprogram: pilchard herpesvirus infection in wild pilchards. Fisheries Research and Development Fund Corporation Project Number 2002/044. In Draft Crockford M., McColl K., Jones J.B. and Wilcox G.E. Comparison of 3 molecular methods for the detection of Pilchard herpesvirus in archived paraffin-embedded tissue and frozen tissue. Submitted to Diseases of Aquatic Organisms 2007; currently under peer review Whittington R.J., Crockford M., Jordan D. and Jones J.B. Survey for herpesvirus in pilchard, a marine pelagic finfish, in Australia. Accepted for submission in Journal of Fish Diseases 2007 Crockford M., Jones J.B., Hillier P. and Wilcox G.E. Molecular detection of Pilchard herpesvirus in archived pilchard (Sardinops sagax neopilchardus) paraffinembedded tissues from a transmission trial conducted in v

7 Table of contents Declaration Abstract Acknowledgements Publications Abbreviation and definitions i ii iv v ix CHAPTER 1. GENERAL INTRODUCTION 1 CHAPTER 2. REVIEW OF THE LITERATURE INTRODUCTION Location and origin of the PHV epizootics Method of transmission of PHV Possible environmental influences contributing to the epizootics Pathology of the PHV-associated disease Clinical signs of PHV infection Lesions associated with the PHV infection Identification of PHV within the gills of affected pilchards Diagnosis of PHV infection AQUATIC HERPESVIRUSES Channel catfish virus Oncorhynchus masou virus Salmonid herpesvirus Koi herpesvirus Cyprinid herpesvirus Goldfish haematopoietic necrosis virus Ranid herpesviruses Other aquatic herpesviruses OVERVIEW OF HERPESVIRUS BIOLOGY General characteristics of herpesviruses Herpesvirus replication Encapsidation and release of virus particles Latency in herpesviruses 37 vi

8 CHAPTER 3. ATTEMPTS TO OBTAIN ADDITIONAL PILCHARD HERPESVIRUS SEQUENCE DATA SUMMARY INTRODUCTION MATERIALS AND METHODS Extraction of DNA from gill tissue PCR for the detection of PHV pzero DNA library used to obtain additional PHV genomic sequence data PCR for the detection of insert in pzero plasmids Freeze-squeeze method for extraction of DNA from agarose gels Sequencing RESULTS Development of PCR based on the ORF 62 gene Comparison of 373 bp of PHV genomic DNA extracted from tissues of fish in the 1995 and 1998 PHV epizootics Attempt to obtain additional sequence of PHV using pzero DNA library DISCUSSION 50 CHAPTER 4. ATTEMPTED TRANSMISSION OF PILCHARD HERPESVIRUS TO WILD CAUGHT PILCHARDS SUMMARY INTRODUCTION MATERIALS AND METHODS Transmission trial ISH i Riboprobes ii ISH procedure DNA extraction from paraffin-embedded tissues i Preparation of tissue sections ii Extraction of DNA from tissue sections PCR assay Real-time PCR assay RESULTS transmission trial in wild-caught pilchards 61 vii

9 4.4.2 ISH PCR Real-time PCR DISCUSSION 67 CHAPTER 5. EPIDEMIOLOGICAL ASPECTS OF PILCHARD HERPESVIRUS INFECTION SUMMARY INTRODUCTION MATERIALS AND METHODS Fish samples ISH PCR Real-time PCR RESULTS Histological examinations ISH PCR Real-time PCR DISCUSSION 79 CHAPTER 6. A SURVEY OF WILD PILCHARDS FOR THE PRESENCE OF PILCHARD HERPESVIRUS SUMMARY INTRODUCTION MATERIALS AND METHODS Survey design Fish samples Extraction of DNA from gill tissue Real-time PCR RESULTS DISCUSSION 89 CHAPTER 7. GENERAL DISCUSSION 92 BIBLIOGRAPHY 98 viii

10 Abbreviations and definitions Abbreviation Definition AAHL Australian Animal Health Laboratories ATP Adenosine triphosphate BAC Bacterial artificial chromosome bp base pair CCO Channel catfish ovary CCV Channel catfish virus CHV1 Cyprinid herpesvirus 1 cm centimetre CMV Cytomegalovirus CNGV Carp nephritis and gill necrosis virus DAS Downstream activating sequence DBF DAS binding factor DNA Deoxyribonucleic acid E Early (gene) EBV Epstein-Barr virus E.coli Escherichia coli FHL Fish Health Laboratory FV4 Frog virus 4; RaHV2 g times gravity GFHNV Goldfish haematopoietic necrosis virus h hour HEPC Herpesviral epidermal proliferation in carp HSC Herpesvirus septicaemia in carp HSV1 Herpes simplex virus 1 HVEM Herpesvirus entry mediators HVHN Herpesviral haematopoietic necrosis IE Immediate early (gene) ISH in situ Hybridisation IU International unit kbp kilo base pairs kg kilogram KHV Koi herpesvirus km kilometre L Late (gene) LAT Latency-associated transcript LB Luria-Bertani (broth or medium) LTHV Lucke tumour herpesvirus; RaHV1 M molar mg milligram MHC Major histocompatibility complex ix

11 MHV Murine herpesvirus min minute ml millilitre mm millimolar MQ Milli-Q (water) mrna messenger RNA MW Molecular weight ng nanogram nm nanometre nmol nanomole NSW New South Wales NZ New Zealand OMV Oncorhynchus masou virus ORF open reading frame Ori Origin of replication PCR Polymerase Chain Reaction PHV Pilchard herpesvirus pmol picomole RaHV1 Ranid herpesvirus 1; LTHV RaHV2 Ranid herpesvirus 2; FV4 RNA Ribonucleic acid SA South Australia SaHV1 Salmonid herpesvirus 1 SaHV2 Salmonid herpesvirus 2 sec second TIF Transcription initiation factor TK thymidine kinase TNF Tumour necrosis factor U Units of enzyme activity UK United Kingdom µl microlitre UL unique long (region of herpesvirus genome) µm micromolar US unique short (region of herpesvirus genome) USA Unites States of America UV ultra-violet VHS Viral host shutoff (protein) v/v volume per volume VZV Varicella-Zoster virus WA Western Australia WSHV1 White sturgeon herpesvirus 1 WSHV2 White sturgeon herpesvirus 2 x